Minimally Invasive Cell-Free Human Embryo Aneuploidy Testing (miPGT-A) Utilizing Combined Spent Embryo Culture Medium and Blastocoel Fluid -Towards Development of a Clinical Assay

Valeriy Kuznyetsov, Svetlana Madjunkova, Rina Abramov, Ran Antes, Zenon Ibarrientos, Gelareh Motamedi, Afsaneh Zaman, Iryna Kuznyetsova, Clifford L Librach, Valeriy Kuznyetsov, Svetlana Madjunkova, Rina Abramov, Ran Antes, Zenon Ibarrientos, Gelareh Motamedi, Afsaneh Zaman, Iryna Kuznyetsova, Clifford L Librach

Abstract

Preimplantation genetic testing for aneuploidies (PGT-A) using trophectoderm (TE) biopsy samples is labour intensive, invasive, and subject to sampling bias. In this study, we report on the efficacy and factors affecting accuracy of a technique we pioneered for minimally invasive preimplantation genetic testing for aneuploidy (miPGT-A). Our technique uses cell-free embryonic DNA (cfeDNA) in spent embryo culture medium (SEM) combined with blastocoel fluid (BF) to increase the amount of assayable cfeDNA. We compared miPGT-A results (n = 145 embryos) with standard PGT-A analysis of the corresponding trophectoderm biopsy. We found that accuracy of miPGT was not related to blastocyst morphological grade. The overall concordance rate per sample for euploidy/aneuploidy status between miPGT-A and TE biopsy samples was 88/90 (97.8%), and was not different between good 47/48 (97.9%) and moderate/low quality blastocysts 41/42 (97.9%) (p > 0.05). Importantly, we also discovered that for cfeDNA analysis, the SurePlex whole genome amplification (WGA) kit can be utilized without an additional cell lysis/extraction DNA step; this efficiency likely reduces the risk of maternal contamination. Regarding origin of embryonic cfeDNA, the average amount of miPGT-A WGA-DNA we obtained from blastocysts with different morphological grades, as well as the size miPGT-A WGA-DNA fragments, suggest that it is unlikely that apoptosis and necrosis are only mechanisms of DNA release from the inner cell mass (ICM) and TE into BF and SEM.

Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Four examples of NGS results representing 24 chromosome copy number plots from TE biopsy and corresponding miPGT samples with concordant results for aneuploidy in three examples and mosaic-complementary in terms of loss versus gain on chromosome 4q (segmental chromosomal mosaicism) in the fourth embryo.
Figure 2
Figure 2
Example of NGS results from TE biopsy and corresponding miPGT-1 (WGA with cell lysis) and miPGT-2 (WGA without cell lysis) samples.
Figure 3
Figure 3
Simplified morphological scoring of blastocyst categorized them into two groups: good quality (≥BB) and moderate/low quality (

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